United States Patent (19 11 4,198,344 Ferrari (45) Apr. 15, 1980

54 PROCESS FOR THE PREPARATION OF (56) References Cited POLYHYDROXYLATED STEROIDS, U.S. PATENT DOCUMENTS LYSERGOL AND ERGOLINICALKALOIDS 3,795,686 3/1974 Ferrari et al...... 260/.397.2 3,828,082 8/1974 Canonica et al...... 260/.397.2 75 Inventor: Giorgio Ferrari, Milan, Italy Primary Examiner-Elbert L. Roberts Attorney, Agent, or Firm-Cushman, Darby & Cushman 73 Assignee: Simes Societa Italiana Medicinalie Sintetici S.p.A., Milan, Italy (57) ABSTRACT A method is disclosed for extracting lysergol and ergo linic alkaloids from seeds of plants belonging to the 21 Appl. No.: 931,296 Convulvulaceae family, Ipomoeae section, petaloidea genus (Choisy). The process applies to ground and 22 Filed: Aug. 4, 1978 defatted seeds and, after an orderly sequence of extrac tion, filtrations and crystallization, a first insoluble frac (30) Foreign Application Priority Data tion is obtained, which contains the expected ergoline alkaloids and lysergol, whereafter a second sequence of Aug. 12, 1977 CH) Switzerland ...... 9916/77 extractive operations is carried out in order to separate as discrete fractions, the ergolinic alkaloids on the one 51) Int. Cl...... C07 9/00 hand, and the lysergol on the other hand. 52 U.S. Cl...... 260/.397.25 58 Field of Search ...... 260/.397.25, 397.2 18 Claims, No Drawings 4,198,344 1 2 In the family of the polyhydroxylated steroids, ecdi PROCESS FOR THE PREPARATION OF sone, that is 2beta, 3beta, 14alpha, 22/R, 25-hexohy POLYHYDROXYLATED STEROIDS, LYSERGOL droxy-5beta-colest-7-en-6-one, having the formula: AND ERGOLINIC ALKALOIDS OH (III) The present invention concerns a process for the extraction of certain substances which belong to the polyhydroxylated steroid class, also known as phytoec disones, and to the ergolic alkaloids class. More specifi cally, the method according to the present invention 10 regards the obtaining of crustecdisone, makysterone A, HO ecdisone, muristerone, lysergol, chanoclavine and other ergoline alkaloids. HO Among the above-mentioned products, the first four 15 belong to the class of the hormones of the metamorpho sis of insects and can be characterized as follows: (l) crustecdisone, that is, 2beta, 3beta, 14alpha, 20/R, was the first to be isolated and identified (A. Butenandt, 22/R, 25-hexahydroxy-5-beta-colest-7-en-6-one, having P. Karlson and Coll., Amm. Chem.662, 1 (1963) ) and 20 represented the starting point research conerning the the formula: properties of such steroids as regards the influence thereof in the biological cycle of insects. Finally, in recent years a new steroid has been identified and ob tained, muristerone, that is, 2beta, 3beta, 5beta, 11alpha, 25 14alpha, 20/R, 22 R-heptahydroxy-5beta-colest-7- en-one having the formula: g H (IV) OH 30

Such hormone was isolated in the past for the first 35 time from a crustacean, Jasus calandei (Chem. Com. 1966, page 37) in the amount of 2 mg of pure hormone OH from 1 ton of crustacea. Its presence has been found also O in Bombyx mori, together with ecdisone. It is furthermore present in the insect Antharea pernyi 40 All such steroids, in their capacity as hormones, con (200 mg from 31 kg of pupa). trolling the metamorphosis of insects, have found prev It has been isolated from the plants of Podocorpus alent application as insecticides, both as such and/or in elatus, impure with sterols (Chem. Com. 1966, page combination. 905), in the amount of 0.05% of the weight of the vege 45 As regards lysergol and chanoclavine, they are ergo tal material (Chem. Com. 1968, page 971): it is also linic alkaloids, especially useful as intermediates in the present in P. macrophyllus (Tele 1968, page 3883). production of drugs, and have long been well-known in It is also present in Polypodium vulgare L. (0.07-1.0% the art. It has now been found, and this forms the main sub dry weight) (Phytochemistry 9, 1247 (1970)). ject matter of the present invention, that polyhydrox Moreover, makisterone A, that is, 2beta, 3beta, 14al 50 ylated steroids and ergolinic alkaloids, particularly pha, 20/R, 22/R, 25-hexahydroxy-24-methyl-5-beta those above-mentioned, are contained in considerable colesten-7-en-6-one, having the formula: amounts in a plant of the Ipomoea class (Convol vulaceae family), from which they can be extracted in

(II) 55 an economically and industrially advantageous way, by means of the method of the present invention. More specifically the method of the present inven tion, which has the purpose of obtaining the hormones of metamorphosis of insects and the above-identified 60 ergoline alkaloids, is characterized by the fact that the extraction is carried out by starting from parts, particu larly seeds, of Ipomoea petaloidea Choisy. The process according to the present invention is thus H characterized by the following operations: O 65 (a) extraction with a solvent of the parts of Ipomoea petaloidea Choisy, in particular seeds, previously pow has been found in Podocorpus macrophyllus in the dered and defatted, such solvent being selected from amount of 0.001% (T.Lett. 1969, page 3887). halogenated aliphatic hydrocarbons and their mixtures 4,198,344 3. 4. with aliphatic alcohols containing from 1 to 4 carbon line precipitate is obtained, which-on thin layer atoms, and a base, such extraction being possibly re chromatography-proves to consist of makisterone A peated several times. and ecdisterone. This precipitate, several times crystal (b) concentration to low volume, at a temperature lized from methanol, provides pure, makisterone A; in lower than 40 C., of the extract, or the combined ex its turn, the hydromethanolic solution left from the tracts, which are then allowed to stand at a low temper separation of the crystalline precipitate (makisterone ature with separation of a precipitate (total crystallizate) A -- ecdisterone) is evaporated to dryness and chro and a supernatant phase; matographed on alumina, thus forming a fraction (c) repeated washing with water of the total crystalli mainly comprising muristerone. zate, thus obtaining an insoluble fraction and an aqueous 10 The residual mother liquors from the crystallization solution; of ecdisone or makisterone A, after evaporation to dry (d) washing with methanol of the insoluble fraction ness, provide two residues which are combined and, by thus obtaining insoluble residue (alkaloidal fraction), simple crystallization from acetone, provide crustecdi which is combined with said supernatant phase, and a sone or ecdisterone. methanolic solution which, combined with said aqueous 15 On the alkaloidal fractions of extraction of the drug, solution, forms the steroidal fraction; on the other hand, separation and isolation of the ergo (e) treatment of the alkaloidal fraction to isolate and linic alkaloids is carried out, utilizing aqueous solutions purify lysergol and chanoclavine; of acids. It has been found that diluted aqueous solutions (f) treatment of the steroidal fraction to isolate the of phosphoric acid prove particularly suitable. The hormones of the metamorphis of insects. 20 extraction of the organic phase with aqueous solutions According to the invention, the seeds of Ipomoea of acids is continued until Ehrlidh's test for alkaloids is petaloidea Choisy are powdered so as to obtain a meal. negative. The powdered drug is subsequently in cold conditions From the collected acid extracts, weakly alkanilized with petrol or light naphta (for ex. 80°-90° C.) in order with a base (preferably aqueous ammonia), the alkaloids to separate all the fats. The operation is repeated to 25 are extracted again with a mixture of a chlorinated complete exhaustion, which is obtained by repeating aliphatic hydrocarbon and an aliphatic alcohol. Particu such operation three or four times. larly suitable is a mixture of chloroform/methanol in The defatted drug then undergoes extraction with the volume ratio 7:3. solvents suitable for removing the active principles. The extractions are repeated until Ehrlich's test Especially useful for such purpose are the aliphatic 30 proves negative. The collected extracts are washed halogenated hydrocarbons such as, for example, chloro with water once, dried on anhydrous sodium sulphate form, carbon tetrachloride, methylene chloride, trichlo and then evaporated to dryness, at a temperature lower roethylene. In order to facilitate the extraction the em than 30 C., under low pressure. The residue proves to ployment of a mixture of the aliphatic halogenated hy be formed of the alkaloidal fraction of Ipomoea petaloi drocarbon with a small percentage of an aliphatic alco 35 dea Choisy and shows when examined by thin layer hol of low molecular weight (1-4 carbon atoms) is pre chromatography, utilizing as solvent methylene chlo ferred. Among the alcohols which are useful for such ride-methanol-benzol (25:5:5), and, as detector, a 3% purpose mention can be made of methyl, ethyl, isopro solution of vanillin in alcohol with 0.5 in volume of pyl alcohol whilst, among bases, ammonia and amines concentrated sulphuric acid, after activation for 5 min are preferred. The added amounts of alcohols can reach 40 utes at 110-120° C., the presence of various ergolinic 15%, whilst, the alkaline bases can reach 5% by volume alkaloids, among which mainly lysergol, and second with respect to the chlorinated hydrocarbon. arily chanoclavine. In the case of the preferred mixture, that is, chloro It has been found that lysergol can be isolated from form-methanol-ammonia, the corresponding volumetric the total alkaloids by simply washing the mass with a ratios are 9:0, 9:0, 1. 45 lower aliphatic alcohol such as methanol, thus forming The extraction is preferably carried out in a tempera the insoluble residue in cold conditions. Such residue, ture range of from 10 to 50° C., whilst the number of after filtration, can be further purified by crystallization extractions necessary for the exhaustion of the drug is from a suitable organic solvent, or from a dimethylsul comprised between three and five. The extraction, in phoxide-water mixture. Lysergol can be particularly each case, is repeated until Liebermann's test for steroid 50 well purified by repeated crystallizations from dime identification is negative. thylsulphoxide-water 1:1. The product obtained in this The combined extracts are evaporated at a tempera way shows the following characteristics: on centesimal ture lower than 40 C. to a tenth of their initial volume analysis it corresponds to the empirical formula and allowed to stand for some days at a temperature of C16H18ON2: C% calc., 75.55; H%, 7.14; N%, 11.02. 0-4 C. A precipitate (total crystallizate) is thus ob 55 C% found, 75.39; H%, 7.22; N%, 10.96. tained, which represents the sterol fraction of the drug, P. M. 254.3; m.p. (crystallized from alcohol) and also the mother liquors containing part of the alka 253-255 C. (with dec.); ap20 = +54 (c=0.3 in pyri loids of the starting drug. The total crystallizate under dine. goes repeated washings with water to an insoluble frac The present invention also provides for the further tion and to an aqueous solution. The water-insoluble 60 isolation of chanoclavine from the residue containing fraction, still wet, is washed with methanol, thus leading the total ergolinic alkaloids after the isolation of lyser to an insoluble fraction, formed by the main part of the gol. For such purpose, the present invention provides alkaloid fraction of the drug. The methanolic solution is for the following further operations: in turn evaporated to dryness and the residue, repeat (a) evaporating to dryness in vacuo and at tempera edly crystallized from water, leads to pure ecolisone. 65 tures lower than 60 C., the residual mother liquors The aqueous solution obtained from the washings of from the extraction of lysergol; the total crystallizate is, instead, mixed with the same (b) dissolving the residue in pyridine in excess and volume of methanol. After allowing to stand, a crystal adding acetic anhydride in an amount by weight equal 4,198,344 5 6 to that of the dry residue, allowing the mixture to stand The organic extracts are washed once with water, for 24 hours; then dried on anhydrous sodium sulphate and evapo (c) precipitating crude chanoclavine diacetate by rated to dryness, under low pressure at a temperature pouring the reaction product thus obtained over an lower than 30° C. excess of water and ice; and 5 The residue (200-270 g) formed by the total ergolinic (d) purifying the crude chanoclavine diacetate after alkaloids of the drug has the composition indicated in drying by recrystallization from ethyl acetate. the introductory part of this specification and is now The thus obtained chanoclavine diacetate shows the agitated in cold conditions with 500 g of methanol for 1 following characteristics: hour. The insoluble part is filtered and dried in vacuo. m.p. 172-173° C.-ap20-53.4 (c=0.95 in pyri- 10 Further purification of the insoluble fraction (lyser dine) gol) is carried out by dissolving the crude product in an ap20-58 (c= 1% in chloroform)-I.R. spectrum equal amount of dimethylsulphoxide slightly heating in in CHCl3, characteristic absorption bands, at 1625 a water bath. The solution is treated with decolourizing cm-1 (N-COCH3); 1730 cm-l (O-COCH3)-U.V. carbon, carefully filtered and to the filtrate there is spectrum in MeOH----1% CHCl3: Anax 283 nm (log 15 added the same volume of distilled water. Then it is left e3.81); 216 nm (log e4.76): 291 nm (log e3.78) under crystallization. The crystallized product is sepa centesimal analysis for C20H24O3N2 (340.4) calc. C% rated by filtration and dried in vacuo to constant 70,6; H% 7.1; N% 8.2. found 70.37.158.3. weight. As regards the steps of the above identified process The operation is repeated to obtain a product having the following should be noted: 20 the characteristics indicated in the introductory part. (1) the dry residue obtained by evaporation of the mother liquors collected from the extraction of lyser EXAMPLE 2 gol, is weighed and dissolved, preferably in about three The methanolic mother liquors obtained from the times its weight of anhydrous pyridine; washings of the total ergolinic alkaloids identified in (2) the excess of water and ice for the precipitation of 25 EXAMPLE 1, are placed in a rotary evaporator and the the chanoclavine diacetate is about ten times the volume solvent is completely removed in vacuo to dryness at a of the reaction product in step (b); temperature lower than 60° C. The residue (70–90 g) is (3) the crude chanoclavine diacetate precipitate is taken up with anhydrous pyridine (210-270 ml) and separated by filtration after repeated washings with (70-90 ml) acetic anhydride is added to the solution. water and then purified. 30 The mass, protected from humidity, is allowed to stand The following examples are provided, solely to illus for 24 hours. Subsequently it is poured onto ice and trate the invention without in any way limiting it: water (2000–2200 ml). The separated product is allowed to decant and repeatedly washed with water. It is fil EXAMPLE 1. tered in vacuo, dried and recrystallized from ethyl ace 40 kg of seeds of Ipomoea petaloidea Choisy are 35 tate (30-45 g). The chanoclavine diacetate thus ob ground in a mill in order to obtain a meal with particle tained proves pure, when examined by thin layer chro size comprised between 40 and 60 mesh per cm. matography on Kieselgel GF254 (Type 60), using meth The ground drug is exhausted in cold conditions in an ylene chloride-benzene-methanol 25:5;5 as solvent, and extractor provided with agitator 5 times, with petrol, vanillin/sulphuric acid as reagent. b.p. 80°-90° C. 150 liters of solvent are employed each 40 time. Each extractor lasts 3 hours, under agitation. The EXAMPLE 3 defatted drug, after complete removal of petrol, is twice 40 kg of seeds of Ipomoea petaloidea Choisy, finely extracted in the same apparatus with a mixture of chlo ground, defatted by threefold agitation with 70 liters of roform-methanol-ammonia=9:0.9:0.l. light petrol (b.p. 30°-60° C). The defatted drug was 160 Liters of mixture are employed. Subsequently the 45 extracted three times with 120 liters of a mixture of mass is extracted a further three times with chloroform CHCl3/MeOH/NH4OH (9:09:0.1), for 12 hours, under alone (100 liters). The collected extracts are concen agitation and at room temperature, and three times with trated at low pressure, at a temperature lower than 30 100 liters of CHCl3; the combined extracts were con C. to a volume of 25 liters. The concentrate is allowed centrated to 1/10 of their volume at 40 C. The solid to stand in a refrigerator at 0-4 C. for two days. The 50 precipitate separating after 4-5 days in a refrigerator, separated solid (total crystallizate) is filtered by pumps was collected, washed with CHCl3 and dried. From the and the panel is dried in vacuo. The dried panel mother liquors, after concentration, further solid depos (300/400 g) is suspended in cold water 10 times its ited after storage in a refrigerator, this precipitate was weight and agitated for 1 hour. The insoluble residue is also collected, washed with CHCl3 and dried. The filtered again, dissolved in methanol, three times its 55 amount of combinated solids (fraction A) was 231.g. weight, and the methanolic solution is added to the The mother liquors contained alkaloids (75 g) and fur main filtrate. ther compounds belonging to fraction A. These latter The filtrate is now washed with six liters of water in were isolated by means of direct extraction with water a separator. The organic phase is then further concen and the residue, after evaporation to dryness (55 g) was trated to a volume of eight liters, in vacuo at a tempera- 60 combined with fraction A (in all, 336 g). ture lower than 30° C. Such concentrate is exhausted by Fraction A was triturated 4 times with water (1500 extracting it four times with a 5% phosphoric acid solu ml) at 25 C. for one hour. The insoluble part (90 g) tion. In all, eight liters of acid solution, are employed. contained the alkaloids, whilst the dissolved product, The collected acid extracts are alkalinized slightly with after evaporation to dryness (fraction B, 250 g) mainly aqueous ammonia to pH 8 and exhausted by extracting 65 contained phytoecdisones. Fraction B was triturated with a mixture of chloroform/methanol=7:3. Four four times with water (1000 ml) at 25 C. for 1 hour. extractions are carried out, and in all eight liters are The combined filtrates, after addition of MeOH, were employed. concentrated under low pressure at 35 C. to about one 4,198,344 7 8 fifth of the initial volume, allowed to crystallize in an negative Ehrlich's test for alkaloids; from the combined ice bath, collecting the crystalline fraction C (50 g). acid extracts, slightly alkalinized with a base, the alka From fraction C, after dissolution in methanol contain loids are re-extracted with a mixture of a chlorinated ing 1% water, crystals of reasonably pure makisterone aliphatic hydrocarbon and an aliphatic alcohol, continu A, 10 g, deposited, whilst crustecdisone remained in the 5 ing such re-extraction until there is a negative Ehrlich's mother liquors from which it was obtained by means of test, and the combined extracts, washed only once with evaporation of the solvent (Residue R). The residual water and dried on anhydrous sodium sulphate are mother liquors from precipitation of crystalline fraction evaporated to dryness at a temperature lower than 30 C were evaporated to dryness (110 g) and chromato C. under low pressure, thus forming a residue compris graphed on silica gel column (Merck, 1000 g), collect- 10 ing the alkaloidal fractions of the extracted plant ing and combining the fractions eluted with EtOAc. wherein lysergol is mainly present. After evaporating to dryness, such fractions were dis 5. A process according to claim 4, characterized by solved in EtOAc and left to crystallize, thus providing the fact that said acid aqueous solution is a phosphoric muresterone (10 g). From the subsequent fractions acid solution. eluted with a mixture of EtOAc and 10% methanol, 15 6. A process according to claim 4, characterized by crustecdisone was obtained (20g). the fact that said extraction mixture is a mixture of The water-insoluble part of fraction B, still wet after chloroform and methanol in volume ratio 7:3. the treatment in water, was triturated with methanol 7. A process according to claim 4, characterized by (200 ml) at 50° C. The insoluble part still contained the fact that lysergol is isolated from said residue by alkaloids (9.5 g) whilst the soluble part D after evapora- 20 washing with lower aliphatic alcohol and further puri tion to dryness at a temperature lower than 35 C., was fied by crystallization with an organic solvent or a mix repeatedly crystallized from water providing ecdisone ture of dimethylsulphoxide and water, preferably in the (5 g) as a solid, whilst the mother liquors contained ratio 1:1. further crustecdisone, which was obtained by means of evaporation to dryness and crystallization (Residue R2). 25 8. A process for the extraction of ergolinic alkaloids The two combined residues R1 and R2 were recrystal according to claim 4, characterized by the fact that the lized from metallic carbonate thus providing relatively mother liquors, which are residual from the lysergol pure crustecdisone (60 g). isolation step, are evaporated to dryness in vacuo and at Finally, the alkaloidal part of the extract in EXAM a temperature lower than 60 C., the dry residue is PLE 3 was treated as in EXAMPLES 1 and 2 to re- 30 dissolved with an excess of anhydrous pyridine prefera cover lysergol and chanoclavine whilst the steroidal bly about three times the weight of the dry residue and fraction of EXAMPLE 1 was treated as in EXAMPLE acetic anhydride is added in an amount equal to that of 3 to recover each single phytoecdisone. the residue, the reaction mixture is left to stand for 24 What I claim is: hours; chanoclavine diacetate is precipitated by pouring 1. In a process for the production of hormones of 35 the mixture produced by the reaction into an excess of themetamorphosis of insects and for the production of water and ice, preferably about 10 times in volume over ergolinic alkaloids the improvement comprising ex the volume of the reaction product; crude chanoclavine tracting said hormones and alkaloids from a plant of the diacetate is filtered and purified, after crystallization, by Convolvulaceae family, Ipomoeae section, Petaloidea recrystallization from ethyl acetate. (Choisy) genus. 40 9. A process according to claim 1, for the extraction 2. A process according to claim 1, characterized by of pure polyhydroxylated steroids, characterized by the the fact that the extraction is performed on the seeds, fact that from said methanolic solution, after evapora ground and previously defatted, with a chlorinated tion to dryness, a residue is obtained, which, after re aliphatic hydrocarbon solvent, in a temperature range peated crystallizations from water, provides pure ecdi of from 10 to 50° C. and repeating the extraction 3-5 45 sone and mother liquors. times. 10. A process according to claim 1 for the extraction 3. A process according to claim 2, characterized by of polyhydroxylated steroids, characterized by the fact the fact that to said solvent there is added a small that said aqueous solution is treated with methanol in a amount, not more than 15% by volume over the solvent ratio of 1:1, with separation of a crystallizate, or insolu itself, of an aliphatic alcohol of low molecular weight 50 ble phase, containing molesterone A and crustecdisone (having from 1 to 4 carbon atoms) and a small amount, and of mother liquors containing murosterone. not more than 5% by volume over the solvent, of a base 11. A process according to claim 1, characterized by selected from ammonia and amine. the fact that from said crystallizate or insoluble phase 4. A process according to claim 2, characterized by pure makisterone A is isolated by repeated crystalliza the fact that the extracts, which have been combined 55 tions from methanol, thus providing a hydroalcoholic and concentrated to a tenth of their initial volume at a solution containing ecdisterone. temperature lower than 30° C. and under low pressure, 12. A process according to claim 1, characterized by are left to stand for some days at 0-4 C. and subse the fact that said hydroalcoholic solution and said resid quently filtered off, thus obtaining a total crystallizate ual mother liquors from the separation of ecdisone are and mother liquors, said total crystallizate being washed 60 evaporated to dryness and from the combined residues, with water, with separation of an insoluble residue and by crystallization from acetone, they provide ecdister an aqueous solution, said insoluble residue being washed one or crustecdisone. with methanol with separation of an insoluble phase and 13. A process according to claim 1, characterized by a methanolic solution, said insoluble phase together the fact that from said mother liquors containing muris with said mother liquors undergoing separation of the 65 terone, by evaporation to dryness and chromatography ergolinic alkaloids by means of extraction with an acid on alumina, muristerone is obtained. aqueous solution in order to separate the ergolinic alka 14. A process according to claim 8 wherein the alka loids, said extraction being continued until there is a loid extracted is chanoclavine. 4,198,344 9 10 15. A process according to claim 9 wherein the ste- from the group consisting of crustecdisone, makisterone roid extracted is pure ecdisone. A, ecdisone, muristerone, lysergol and chanoclavine. 16. A process according to claim 2 wherein the sol- 18. A process according to claim 17 wherein the vent is chloroform, carbon tetrachloride, methylene solvent is selected from the group consisting of chloro chloride or trichloroethylene. 5 form, carbon tetrachloride, methylene chloride and 17. A process according to claim 1 wherein the hor- trichloroethylene. mones and ergolinic alkaloids produced are selected k is sk k

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